JPWO2018234506A5

JPWO2018234506A5 -

Info

Publication number: JPWO2018234506A5
Application number: JP2019570982A
Authority: JP
Publication date: 2023-07-05
Anticipated expiration: 2038-06-21

Description

別の側面では、組換えポックスウイルスによりコードされる前記１以上のネオペプチドの各々は、１以上の腫瘍特異的突然変異を含んでなり、好ましくは、前記ネオペプチドの少なくとも６０％が、ミスセンスまたはフレームシフト突然変異を含んでなる。望ましくは、１以上のネオペプチドが、１６～９０アミノ酸残基、好ましくは、１７～８５アミノ酸残基、より好ましくは、１８～８０アミノ酸残基の長さを有する。好ましい実施形態では、ミスセンス突然変異を含んでなるネオペプチドは、１８～２９残基の長さを有し、フレームシフト突然変異を含んでなるネオペプチドは、３０～８０アミノ酸残基の長さを有する。ミスセンス突然変異を有するネオペプチドの少なくとも８０％が、中心の位置で置換アミノ酸を有する。好ましい実施形態では、いくつかのネオペプチド、好ましくは全てのネオペプチドが、１以上の融合体の形態で組換えポックスウイルスにより発現される。組換えポックスウイルスは、好ましくは、自殺遺伝子および免疫賦活遺伝子からなる群から選択される１以上の治療遺伝子をさらにコードし得る。 In another aspect, each of said one or more neopeptides encoded by the recombinant poxvirus comprises one or more tumor-specific mutations, preferably at least 60% of said neopeptides are missense or comprising a frameshift mutation. Desirably, the one or more neopeptides has a length of 16-90 amino acid residues, preferably 17-85 amino acid residues, more preferably 18-80 amino acid residues. In preferred embodiments, the neopeptide comprising the missense mutation has a length of 18-29 residues and the neopeptide comprising the frameshift mutation has a length of 30-80 amino acids. have. At least 80% of neopeptides with missense mutations have the substituted amino acid at the central position. In a preferred embodiment, some, preferably all neopeptides are expressed by the recombinant poxvirus in the form of one or more fusions. The recombinant poxvirus may preferably further encode one or more therapeutic genes selected from the group consisting of suicide genes and immunostimulatory genes.

相同組換えは、その欠失ＩＩＩ内にｍＣｈｅｒｒｙ蛍光タンパク質をコードする遺伝子を含有する親ＭＶＡ（ＭＶＡｍＣｈｅｒｒｙ）を用いて、行った。ＭＶＡｍＣｈｅｒｒｙの利点は、最初の開始ＭＶＡｍＣｈｅｒｒｙウイルス（親ウイルス）に感染している細胞から、発現カセットを組み込むことに成功している組換えウイルスに感染している細胞を区別することである。実際に、欠失ＩＩＩ内での発現カセットの組換えが成功している場合は、ｍＣｈｅｒｒｙ遺伝子は除去され、ウイルスプラークは白色として出現する。 Homologous recombination was performed with the parental MVA (MVA mCherry) containing the gene encoding the mCherry fluorescent protein within its deletion III. The advantage of MVA mCherry is that it distinguishes cells infected with a recombinant virus that successfully integrates the expression cassette from cells infected with the original starting MVA mCherry virus (parental virus). Indeed, if the expression cassette has been successfully recombined within deletion III, the mCherry gene will be removed and viral plaques will appear white.

まとめると、これらの結果は、リンカーの非存在は、ＣＥＦ細胞において生じる白色プラークの割合、従って組換えＭＶＡの作製を負に変えるものの、免疫原性に対する大きな影響は及ぼさないことを示す。従って、ベクター構築物の設計を促進するため、従って、特に、１０を超えるネオペプチドの発現が考えられるが、作製される組換えポックスウイルスの割合を減少させるリスクにある構築物において、有害な相同組換え事象のリスクを低減させるために、必要に応じて、リンカーを省略してもよい。 Taken together, these results indicate that the absence of the linker negatively alters the proportion of white plaques produced in CEF cells and thus the production of recombinant MVA, but does not significantly affect immunogenicity. Therefore, to facilitate the design of vector constructs, therefore, deleterious homologous recombination, especially in constructs where the expression of more than 10 neopeptides is possible, but at risk of reducing the proportion of recombinant poxviruses produced. Linkers may be omitted if desired to reduce the risk of events.

Claims

Personalized cancer vaccine comprising a recombinant poxvirus encoding one or more neopeptides, some neopeptides in the form of one or more fusions of 2 to 15 neopeptides, said recombinant A method of preparing said personalized cancer vaccine expressed by a poxvirus, wherein said fusion does not contain any TM segment and exhibits a hydropathy score of 0.1 or less, comprising:
said method comprising an identifying step of identifying one or more neopeptides suitable for being encoded by a recombinant poxvirus;
said one or more neopeptides comprise one or more tumor-specific mutations;
Said identifying step comprises the following sub-steps a) to d), h) and i):
a) extracting DNA from tumor and non-tumor samples;
b) selecting a target region;
c) sequencing said target region from said extracted DNA;
d) identifying one or more tumor-specific mutations by comparing DNA sequences obtained from said tumor sample and said non-tumor sample;
h) Predict the presence of a potential TM segment in the neoepitope itself and a potential TM segment in a neopeptide fusion comprising several neopeptides, and potential neopeptides and/or no TM segments. selecting potential neopeptide fusions that do not contain a TM segment; and i) ranking said potential neopeptides and/or said potential neopeptide fusions by degree of hydrophobicity, said neopeptide fusions selecting the neopeptide and/or neopeptide fusion such that the body exhibits a global hydropathy score of 0.1 or less.

2. The method of claim 1 , wherein the target region is the entire coding region of the genome.

3. The method of claim 1 or 2 , wherein said tumor sample is a tumor biopsy and said non-tumor sample is a biological fluid, cytological material or biopsy.

Said identification step comprises the following further sub-steps e)-g):
e) ranking potential neopeptides by their level of expression in tumors, either at the level of mRNA transcription or at the level of protein translation;
f) selecting a non-self-expressing tumor-specific mutation, wherein said neopeptide is not part of another protein expressed in normal cells of the subject;
The method according to any one of claims 1 to 3 , further comprising one or more of the steps of g) predicting the immunocompetence of the neoepitopes contained in said neopeptide.

The identification step includes
sub-step f),
sub-steps e) and f),
5. The method of claim 4 , further comprising sub-steps f) and g), or all of sub-steps e)-g ) .

A method according to any one of claims 1 to 5 , wherein said method further comprises the step of producing said recombinant poxvirus.

the one or more neopeptide-encoding nucleic acid molecules inserted into the genome of the recombinant poxvirus are placed in one or more expression cassettes under the control of suitable regulatory elements allowing expression in a subject; 7. The method of claim 6 .

said nucleic acid molecule or expression cassette encoding said one or more neopeptides is inserted into the genome of a parental poxvirus to produce said recombinant poxvirus, said parental poxvirus comprising a nucleic acid molecule encoding a neopeptide or 8. A method according to claim 6 or 7 , comprising a cloned fluorescent reporter gene at the insertion site selected for the expression cassette.

9. The method of claim 8 , wherein said fluorescent reporter is selected from the group consisting of GFP (green fluorescent protein), eGFP (enhanced green fluorescent protein), AmCyanl fluorescent protein and mCherry.

Said step of generating said recombinant poxvirus is the further step of cleaving with an endonuclease capable of producing at least one double-stranded break in the nucleotide sequence of said fluorescent reporter, said endonuclease said poxvirus 10. A method according to claim 9 , comprising said further step not cutting the genome of.

Said endonucleases are zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), clustered regularly arranged short palindromic repeats (CRISPR)/Cas9 nucleases and fluorescent reporter genes unique 11. The method of claim 10 , selected from the group consisting of restriction enzymes having a cleavage site.

3. The step of producing the recombinant poxvirus enables producing the recombinant poxvirus in up to 4 weeks with a ratio of recombination to parent comprised between 2 and 0.05. 12. The method according to any one of 6 to 11 .

The method further comprises a manufacturing step, said manufacturing step comprising amplifying to a suitable scale in a suitable producer cell and recovering the produced recombinant poxvirus from the cell culture. The method according to any one of claims 1 to 12 , comprising:

14. The method of claim 13 , wherein said manufacturing step further comprises purifying said harvested recombinant poxvirus.

a) said producer cells are chicken embryo fibroblasts (CEF);
b) said step of recovering the recombinant poxvirus produced comprises a lysis step in which said producer cell membrane is disrupted; and/or
c) A method according to claim 13 or 14 , wherein said purification step comprises a tangential flow filtration (TFF) step.

16. The method of any one of claims 13-15 , wherein the manufacturing process reaches a production of at least ¹⁰⁹ pfu for the personalized cancer vaccine.